Potential role of selected lichens collected from India as antifungal agents against dermatophytes

Fungal infections which include Dermatophytosis are one among the most common communicable disease in the world. Dermatophytes are a group of fungi which cause infection on skin, hair and nails and these fungi do not infect mucosal surfaces. The study was aimed at evaluating the antifungal activity of diethyl ether, methanol and acetone extract of selected lichens Usnea perplexans Stirton, Usnea spinocula Stirton, Usnea subsordiata Stirton, Ramalina conduplicans Vainio, Roccella montagnei Bel emend, Aswath against dermatophytes, Aspergillus flavus and Candida albicans. The invitro antifungal activity was performed by disk diffusion method and broth tube dilution method. All the three extracts of the selected lichens showed antifungal activity against Dermatophytes, Aspergillus flavus and Candida albicans. The activity varied depending upon the lichen and the extract.

Original Research Article https://doi.org/10.20546/ijcmas.2019.804.078

Potential Role of Selected Lichens Collected from India as Antifungal

Agents against Dermatophytes N.S Ravichandran * and S Daya Pauline

Department of Microbiology Sree Mookambika Institute of Medical Sciences,

Kulasekharam, Tamil Nadu, India

*Corresponding author

A B S T R A C T

Introduction

Superficial fungal infections particularly

those involving the skin and mucosal surfaces

constitute a serious problem, especially in

tropical and subtropical developing countries

(1) Dermatophytes have been reported to be

potentially pathogenic (2) and are directly

connected with the skin fungal infections

They are a group of closely related

keratinophilic fungi that invade keratinized

humans and animal tissues such as skin, hair

and nails causing Dermatophytosis

Dermatophytes consist of three genera

Trichophyton, Microsporum, and

Epidermophyton (3) Unlike other fungal

infections, cutaneous mycosis has been considered important in which host immune responses are highly evoked resulting in severe pathologic changes, which are extended deeper into epidermis as well as hair and nails Such fungal infections are mainly

caused by Microsporum species (4) Humid

weather, over population and poor hygienic condition induce the growth of dermatophytes and thereby dermatophytosis

The drugs that are in use to cure dermatophytosis exhibit several side effects and have limited efficiency (5) Further with allopathic drugs, the problem of drug resistance and drug toxicity cannot be ruled

International Journal of Current Microbiology and Applied Sciences

ISSN: 2319-7706 Volume 8 Number 04 (2019)

Journal homepage: http://www.ijcmas.com

Fungal infections which include Dermatophytosis are one among the most common communicable disease in the world Dermatophytes are a group of fungi which cause infection on skin, hair and nails and these fungi do not infect mucosal surfaces The study was aimed at evaluating the antifungal activity of diethyl ether, methanol and acetone

extract of selected lichens Usnea perplexans Stirton, Usnea spinocula Stirton, Usnea

subsordiata Stirton, Ramalina conduplicans Vainio, Roccella montagnei Bel emend, Aswath against dermatophytes, Aspergillus flavus and Candida albicans The invitro

antifungal activity was performed by disk diffusion method and broth tube dilution method All the three extracts of the selected lichens showed antifungal activity against

Dermatophytes, Aspergillus flavus and Candida albicans The activity varied depending

upon the lichen and the extract

K e y w o r d s

Dermatophytosis,

antifungal activity,

Lichen extracts

Accepted:

07 March 2019

Available Online:

10 April 2019

Article Info

out Therefore, there is a distinct need for the

discovery of new, safer and more effective

antifungal agents

The hunt for novel natural bioactive

compounds as a foundation to a new drug

discovery is getting greater awareness as

formerly reliable standard drugs become less

effective against the emerging new strains of

multi drug-resistant pathogens (6) The

challenge for today’s pharmaceutical industry

lies in this discovery of such natural bioactive

compounds and its development (7) Plants

and their preparations have been used as

medicines against infectious diseases Similar

to higher plants, lichens were used since

antiquity as natural drugs

The history of lichens and its biological

activity date back from the period after World

War II Lichens, together with some marine

organism and frog venom, are important

sources of biologically active compounds (8)

These organisms produce secondary

metabolites and many of them are known for

presenting biological and/or pharmacological

activities About 350 secondary metabolites

have been known from lichens and around

200 have been subjected to isolation and

characterization Out of all the secondary

compounds extracted from lichens, the best

known is Usnic acid, an antibiotic with

phenol structure (9) Lists of the antibacterial

and antifungal activities of lichen compounds

and lichens against bacteria and fungi can be

found in a review and a book (10, 11)

In this study we have evaluated the The

selected lichen extracts (Acetone, Methanol

and ethanol) produced antifungal activities

against Aspergillus flavus, Candida albicans

Microsporum gypseum, Trichophyton

mentagrophytes, and Epidermophyton the

most common aetiological agent of

dermatophytosis

To evaluate the antifungal activity of certain

lichen extracts of Usnea perplexans Stirton, Usnea spinocula Stirton, Usnea subsordiata Stirton, Ramalina conduplicans Vainio, Roccella montagnei Bel emend, Aswath against dermatophytes and Candida albicans

Materials and Methods Study design: Descriptive study

Study Setting: Department of Microbiology Sree Mookambika Institute of Medical Sciences, Kulasekharam

Collection of lichens

Lichens specimens were collected from Kodayar of Western Ghats, Kanyakumari District, during summer season Identification

of collected lichens collected lichens were sent to NBRI Lucknow and identified by Dr Upreti

Preparation of the extracts of lichens

Collected lichens were cleaned of extraneous material, dried at room temperature and ground into a coarse powder 100gms of the coarse powder was subjected to successive extraction with the three solvents namely diethyl ether (non polar), acetone (mid polar) and methanol (highly polar) by hot percolation method The extractions were carried out for a period of 72 hours at a temperature not exceeding the boiling point of the solvents At the end of the extraction, the respective solvents were concentrated by evaporation under reduced pressure The crude extracts were then transferred to small glass bottles and placed in a desiccator containing fused calcium chloride These crude extracts were redissolved in respective solvents and the antifungal activity was carried

Preliminary phytochemical analysis of

selected Lichens extract

All the three extracts of the selected were

subjected to preliminary phytochemical

analysis for detection of secondary

metabolites following standard procedures

Antifungal susceptibility test

In-vitro susceptibility testing was done for all

fungal isolates to look for their antifungal

susceptibility This was done by standard

broth dilution technique (NCCLS M-38A,

2002)

Preparation of fungal inoculum

Three dermatophytic fungi were taken for this

study Dermatophytic fungi taken were

Trichophyton mentagrophytes, Trichophyton

rubrum, Microsporum canis, Microsporum

gypseum and Epidermophyton floccosum The

selected standard dermatophytes were grown

on sabouraud dextrose agar (SDA) Twenty

one day old culture of dermatophytic fungi

was scraped with sterile scalpel and

macerated with 10ml of sterile distilled water

The suspension was adjusted

spectrophotometrically to an absorbance of

0.600 at 450mm This suspension was used as

inoculum for MIC and MFC Each tube with

media was inoculated with 20µl of fungal

suspension

Determination of Minimal inhibitory

concentration (MIC)

Susceptibility testing was determined by broth

macro dilution method MIC was analyzed by

incorporating various concentrations of lichen

extracts The SD broth was prepared in a tube

The plant leaf extracts or fractions were

diluted serially with dimethyl sulfoxide

(DMSO) The diluted extracts were added to

the SD broth and made up to 2 ml volume

20µl fungal inoculums were added to the extract containing broth Positive controls were also maintained with known antifungal agents Negative control was maintained with DMSO All the tubes were incubated at 30oC and were read at every two-day up to 14 days

of incubation The MICs were determined by visual observation for the inhibition of growth and were compared with that of the control (positive and negative) tubes

Results and Discussion

Antifungal susceptibility testing is a dynamic field of medical mycology (12) Development and standardization of antifungal susceptibility tests have shown remarkable progress in the field of medical mycology This is the first report being presented on the antifungal activity of Lichens collected from Western Ghats The intensity of the antifungal activity was dependent upon the sort of the extract, its concentration and the tested microorganism Similar differences were also noticed by other investigators (13) Preliminary phytochemical analysis tabulated

in table 1 showed the presence of Phenols, reducing sugars, flavonoids, Saponin, quinones and tannins in all the three extracts

of the selected lichens In addition to these phytoconstituents, Steroids were found in all the lichens extract except in the acetone

extract of Usnea subsordiata stirton and Ramalina conduplicans Vainio The ethanolic extract of Usnea perplexans Stirton did not

showed the presence of glycosides while all the other solvent extracts of the selected lichens showed glycoside presence Alkaloid was also present in all the three extracts of the selected lichens with the exception of

methanolic extract of Ramalina conduplicans Vainio

Antifungal activity of the selected lichens was examined qualitatively and was assessed by the presence or absence of inhibition by disc

diffusion method for Candida albicans and

macro broth dilution method for all the tested

fungi (Table 2) Amphotericin was used as

standard at a concentration of 5mg/ml The

antifungal assay revealed that the selected

lichens showed a potent antifungal activity in

a concentration dependent manner The

antimicrobial activity of ethanolic extract of

the Indian Usnea subsordiata stirton collected

from Western Ghats showed minimum MIC

20 against Candida albicans and A.flavus and

maximum MIC 320 against E floccosum

Methanolic extract of the same showed

minimum MIC 160 against T mentagrophytes

and E floccosum and maximum MIC 640

against Candida albicans Acetone extract of

Usnea subsordiata stirton showed minimum

MIC 20 against E floccosum and Candida

albicans, maximum MIC 320 against

A.flavus, T mentagrophytes, M gypseum

The antimicrobial activity of the ethanolic and

methanolic extract of Usnea perplexans

Stirton showed minimum MIC 160 against

A.flavus T mentagrophytes and E floccosum

A maximum MIC of 320 against M gypseum

and Candida albicans was observed in the

ethanolic extract of Usnea perplexans Stirton

while the methanolic extract of Indian Usnea

perplexans Stirton maximum MIC 640

against M gypseum Acetone extract of

Usnea perplexans Stirton showed minimum

MIC 160 against E floccosum maximum MIC

640 against M gypseum The ethanolic

extract of Indian Usnea spinocula Stirton

showed minimum MIC 160 against all the

tested fungi Methanolic extract of Indian

Usnea spinocula Stirton showed minimum

MIC 160 against T mentagrophytes and E

floccosum, M gypseum and, A.flavus

maximum MIC 640 against C albicans

Acetone extract of Indian Usnea spinocula

Stirton showed minimum MIC 160 against E

floccosum and M gypseum, maximum MIC

320 against T mentagrophytes, A.flavus and

C albicans

Ethanolic extract of Ramalina conduplicans Vainio showed minimum MIC 160 against T mentagrophytes and E floccosum, maximum MIC 320 against A.flavus, M gypseum and C albicans Methanolic extract of Indian Ramalina conduplicans Vainio, showed minimum MIC 160 against E floccosum and, A.flavus maximum MIC 320 against T mentagrophytes, M gypseum and C albicans

Acetone extract of Indian Ramalina conduplicans Vainio, showed minimum MIC

160 against E floccosum and M gypseum,

maximum MIC 320 against T mentagrophytes, A.flavus and C albicans

The antimicrobial activity of ethanol extract

of Indian Roccella montagnei Bel emend, Aswathi showed minimum MIC 160 against A.flavus and E floccosum and M gypseum

maximum MIC 320 against, T mentagrophytes and C albicans Methenolic extract of Indian Roccella montagnei Bel emend, Aswathi, showed minimum MIC 160 against T mentagrophytes and E floccosum and, maximum MIC 320 against A.flavus, M gypseum and C albicans Acetone extract of Indian Roccella montagnei Bel emend, Aswathi showed minimum MIC 160 against

E floccosum, maximum MIC 320 against A.flavus T mentagrophytes, M gypseum and

C albicans

Pure Usnic acid showed minimum MIC 160

against E floccosum and maximum MIC of

320 in the rest of the other tested fungi Usnic acid is one of the most common and investigated lichen compounds Its antimicrobial, antiprotozoal, antiviral, antiproliferative, anti-inflammatory, analgesic, antipyretic, and anti-tumour activities as well as some other properties such as UV protection, allergen, and toxicity have been summarized in two recent reviews Among the lichen substances, the most widely distributed and the most extensively investigated one is Usnic acid (14, 15)

Table.1 Preliminary phytochemical analysis of the selected lichens Lichens

Reducing Su

Usnea subsordiata

stirton

Usnea perplexans

Stirton

Usnea spinocula

Stirton

Ramalina

conduplicans

Vainio

Roccella

montagnei Bel

emend, Aswathi

+ - Present; - - Absent; E – Ethanol; M- Methanol; A- Acetone

Table.2 Antifungal activity of ethanol, methanol and acetone extract of selected lichens

Name of the

lichens

Epidermophyton

Microsporum gypseum

C.albicans

Usnea

subsordiata

stirton

Usnea

perplexans

Stirton

Usnea

spinocula

Stirton

Ramalina

conduplicans

Vainio

Roccella

montagnei Bel

emend,

Aswathi

E – Ethanol; M- Methanol; A- Acetone

After the evaluation of the results, the acetone

extract of Usnea subsordiata stirton showed the

highest inhibitory activity even at minimum

MIC among the other lichen extracts against the

tested fungi It may due to the presence of the

Usnic acid in the selected lichens which showed

a potent antifungal activity

In conclusion, the above results demonstrate

that the antifungal activity of the selected

lichens against dermatophytes, C albicans and

A flavus correlates with the earlier reports on

the antimicrobial activity of the lichens and

Usnic acid, the active component widely

distributed in lichens The obtained results

showed that the tested lichen extracts and

lichens acids showed a significant antimicrobial

activity relative to the tested fungi, which could

be of significance in therapy human, animal and

plant diseases Further studies should be done to

search new compounds from lichens that exhibit

strong antimicrobial activity

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How to cite this article:

Ravichandran, N.S and Daya Pauline, S 2019 Potential Role of Selected Lichens Collected from

India as Antifungal Agents against Dermatophytes Int.J.Curr.Microbiol.App.Sci 8(04): 721-726